The present invention relates generally to diseases in which altered mitochondrial function, such as free radical mediated oxidative injury, leads to tissue degeneration and, more specifically, to compositions and methods for detecting predisposition to such diseases by quantifying extramitochondrial DNA.
A number of degenerative diseases are thought to be caused by or be associated with alterations in mitochondrial function. These diseases include Alzheimer""s Disease, diabetes mellitus, Parkinson""s Disease, Huntington""s disease, dystonia, Leber""s hereditary optic neuropathy, schizophrenia, and myodegenerative disorders such as xe2x80x9cmitochondrial encephalopathy, lactic acidosis, and strokexe2x80x9d (MELAS), and xe2x80x9cmyoclonic epilepsy ragged red fiber syndromexe2x80x9d (MERRF). Other diseases involving altered metabolism or respiration within cells may also be regarded as diseases associated with altered mitochondrial function.
Functional mitochondria contain gene products encoded by mitochondrial genes situated in mitochondrial DNA (mtDNA) and by extramitochondrial genes not situated in the circular mitochondrial genome. The 16.5 kb mtDNA encodes 22 tRNAs, two ribosomal RNAs (rRNA) and only 13 enzymes of the electron transport chain (ETC), the elaborate multi-complex mitochondrial assembly where, for example, respiratory oxidative phosphorylation takes place. The overwhelming majority of mitochondrial structural and functional proteins are encoded by extramitochondrial, and in most cases presumably nuclear, genes. Accordingly, mitochondrial and extramitochondrial genes may interact directly, or indirectly via gene products and their downstream intermediates, including metabolites, catabolites, substrates, precursors, cofactors and the like. Alterations in mitochondrial function, for example impaired electron transport activity, defective oxidative phosphorylation or increased free radical production, may therefore arise as the result of defective mtDNA, defective extramitochondrial DNA, defective mitochondrial or extramitochondrial gene products, defective downstream intermediates or a combination of these and other factors.
Mitochondria are the subcellular organelles that manufacture bioenergetically essential adenosine triphosphate (ATP) by oxidative phosphorylation. Defective mitochondrial activity, including failure at any step of the ETC, may result in the generation of highly reactive free radicals that have the potential of damaging cells and tissues. These free radicals may include reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydroxyl radicals, and potentially other reactive species that may be toxic to cells. For example, oxygen free radical induced lipid peroxidation is a well established pathogenetic mechanism in central nervous system (CNS) injury, such as that found in a number of degenerative diseases, and in ischemia (i.e., stroke).
There are at least two deleterious consequences of exposure to reactive free radicals arising from mitochondrial dysfunction that adversely impact the mitochondria themselves. First, free radical mediated damage may inactivate one or more of the myriad proteins of the ETC. According to generally accepted theories of mitochondrial function, proper ETC respiratory activity requires maintenance of an electrochemical potential in the inner mitochondrial membrane by a coupled chemiosmotic mechanism. Free radical oxidative activity may dissipate this membrane potential, thereby preventing ATP biosynthesis and halting the production of a vital biochemical energy source. In addition, mitochondrial proteins such as cytochrome c and xe2x80x9capoptosis inducing factorxe2x80x9d may leak out of the mitochondria after permeability transition and may induce the genetically programmed cell suicide sequence known as apoptosis or programmed cell death (PCD).
Second, free radical mediated damage may result in catastrophic mitochondrial collapse that has been termed xe2x80x9ctransition permeabilityxe2x80x9d. For example, rapid mitochondrial permeability transition likely entails changes in the inner mitochondrial transmembrane protein adenylate translocase that results in the formation of a xe2x80x9cpore.xe2x80x9d In any event, because permeability transition is potentiated by free radical exposure, it may be more likely to occur in the mitochondria of cells from patients having mitochondria associated diseases that are chronically exposed to such reactive free radicals.
Altered mitochondrial function characteristic of the mitochondria associated diseases may also be related to loss of mitochondrial membrane electrochemical potential by mechanisms other than free radical oxidation, and such transition permeability may result from direct or indirect effects of mitochondrial genes, gene products or related downstream mediator molecules and/or extramitochondrial genes, gene products or related downstream mediators, or from other known or unknown causes.
Diabetes mellitus is a common, degenerative disease affecting 5 to 10 percent of the population in developed countries. The propensity for developing diabetes mellitus is reportedly maternally inherited, suggesting a mitochondrial genetic involvement. (Alcolado, J. C. and Alcolado, R., Br. Med. J. 302:1178-1180 (1991); Reny, S. L., International J. Epidem. 23:886-890 (1994)). Diabetes is a heterogenous disorder with a strong genetic component; monozygotic twins are highly concordant and there is a high incidence of the disease among first degree relatives of affected individuals.
At the cellular level, the degenerative phenotype that may be characteristic of late onset diabetes mellitus includes indicators of altered mitochondrial respiratory function, for example impaired insulin secretion, decreased ATP synthesis and increased levels of reactive oxygen species. Studies have shown that diabetes mellitus may be preceded by or associated with certain related disorders. For example, it is estimated that forty million individuals in the U.S. suffer from late onset impaired glucose tolerance (IGT). IGT patients fail to respond to glucose with increased insulin secretion. A small percentage of IGT individuals (5-10%) progress to insulin deficient non-insulin dependent diabetes (NIDDM) each year. Some of these individuals further progress to insulin dependent diabetes mellitus (IDDM). These forms of diabetes mellitus, NIDDM and IDDM, are associated with decreased release of insulin by pancreatic beta cells and/or a decreased end-organ response to insulin. Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include obesity, vascular pathologies, peripheral and sensory neuropathies, blindness and deafness.
Parkinson""s disease (PD) is a progressive, neurodegenerative disorder associated with altered mitochondrial function and characterized by the loss and/or atrophy of dopamine-containing neurons in the pars compacta of the substantia nigra of the brain. Like Alzheimer""s Disease (AD), PD also afflicts the elderly. It is characterized by bradykinesia (slow movement), rigidity and a resting tremor. Although L-Dopa treatment reduces tremors in most patients for a while, ultimately the tremors become more and more uncontrollable, making it difficult or impossible for patients to even feed themselves or meet their own basic hygiene needs.
It has been shown that the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces parkinsonism in animals and man at least in part through its effects on mitochondria. MPTP is converted to its active metabolite, MPP+, in dopamine neurons; it then becomes concentrated in the mitochondria. The MPP+ then selectively inhibits the mitochondrial enzyme NADH:ubiquinone oxidoreductase (xe2x80x9cComplex Ixe2x80x9d), leading to the increased production of free radicals, reduced production of adenosine triphosphate, and ultimately, the death of affected dopamine neurons.
Mitochondrial Complex I is composed of 40-50 subunits; most are encoded by the nuclear genome and seven by the mitochondrial genome. Since parkinsonism may be induced by exposure to mitochondrial toxins that affect Complex I activity, it appears likely that defects in Complex I proteins may contribute to the pathogenesis of PD by causing a similar biochemical deficiency in Complex I activity. Indeed, defects in mitochondrial Complex I activity have been reported in the blood and brain of PD patients (Parker et al., Am. J. Neurol. 26:719-723, 1989).
Alzheimer""s disease (AD) is a progressive neurodegenerative disorder that is characterized by loss and/or atrophy of neurons in discrete regions of the brain, and that is accompanied by extracellular deposits of xcex2-amyloid and the intracellular accumulation of neurofibrillary tangles. It is a uniquely human disease, affecting over 13 million people worldwide. It is also a uniquely tragic disease. Many individuals who have lived normal, productive lives are slowly stricken with AD as they grow older, and the disease gradually robs them of their memory and other mental faculties. Eventually, they cease to recognize family and loved ones, and they often require continuous care until their eventual death.
There is evidence that defects in oxidative phosphorylation within the mitochondria are at least a partial cause of sporadic AD. The enzyme cytochrome c oxidase (COX), which makes up part of the mitochondrial electron transport chain (ETC), is present in normal amounts in AD patients; however, the catalytic activity of this enzyme in AD patients and in the brains of AD patients at autopsy has been found to be abnormally low. This suggests that the COX in AD patients is defective, leading to decreased catalytic activity that in some fashion causes or contributes to the symptoms that are characteristic of AD.
Focal defects in energy metabolism in the mitochondria, with accompanying increases in oxidative stress, may be associated with AD. It is well-established that energy metabolism is impaired in AD brain (Palmer et al., Brain Res. 645:338-42, 1994; Pappolla et al., Am. J. Pathol. 140:621-28, 1992; Jeandel et al., Gerontol. 35:275, 1989; Balazs et al., Neurochem. Res. 19:1131-37, 1994; Mecocci et al., Ann. Neurol. 36:747-751, 1994; Gsell et al., J. Neurochem. 64:1216-23, 1995). For example, regionally specific deficits in energy metabolism in AD brains have been reported in a number of positron emission tomography studies (Kuhl, et al., J. Cereb. Blood Flow Metab. 7:S406, 1987; Grady, et al., J. Clin. Exp. Neuropsychol. 10:576-96, 1988; Haxby et al., Arch. Neurol. 47:753-60, 1990; Azari et al., J. Cereb. Blood Flow Metab. 13:438-47, 1993). Metabolic defects in the temporoparietal neocortex of AD patients apparently presage cognitive decline by several years. Skin fibroblasts from AD patients display decreased glucose utilization and increased oxidation of glucose, leading to the formation of glycosylation end products (Yan et al., Proc. Nat. Acad. Sci. USA 91:7787-91, 1994). Cortical tissue from postmortem AD brain shows decreased activity of the mitochondrial enzymes pyruvate dehydrogenase (Sheu et al., Ann. Neurol. 17:444-49, 1985) and xcex1-ketoglutarate dehydrogenase (Mastrogiacomo et al., J. Neurochem. 6:2007-14, 1994), which are both key enzymes in energy metabolism. Functional magnetic resonance spectroscopy studies have shown increased levels of inorganic phosphate relative to phosphocreatine in AD brain, suggesting an accumulation of precursors that arises from decreased ATP production by mitochondria (Pettegrew et al., Neurobiol. of Aging 15:117-32, 1994; Pettigrew et al., Neurobiol. of Aging 16:973-75, 1995). In addition, the levels of pyruvate, but not of glucose or lactate, are reported to be increased in the cerebrospinal fluid of AD patients, consistent with defects in cerebral mitochondrial electron transport chain (ETC) activity (Parnetti et al., Neurosci. Lett. 199:231-33, 1995).
Signs of oxidative injury are prominent features of AD pathology and, as noted above, reactive oxygen species (ROS) are critical mediators of neuronal degeneration. Indeed, studies at autopsy show that markers of protein, DNA and lipid peroxidation are increased in AD brain (Palmer et al., Brain Res. 645:338-42, 1994; Pappolla et al., Am. J. Pathol. 140:621-28, 1992; Jeandel et al., Gerontol. 35:275-82, 1989; Balazs et al., Arch. Neurol. 4:864, 1994; Mecocci et al., Ann. Neurol. 36:747-51, 1994; Smith et al., Proc. Nat. Acad. Sci. USA 88:10540-43, 1991). In hippocampal tissue from AD but not from controls, carbonyl formation indicative of protein oxidation is increased in neuronal cytoplasm, and nuclei of neurons and glia (Smith et al., Nature 382:120-21, 1996). Neurofibrillary tangles also appear to be prominent sites of protein oxidation (Schweers et al., Proc. Nat. Acad. Sci. USA 92:8463, 1995; Blass et al., Arch. Neurol. 4:864, 1990). Under stressed and non-stressed conditions incubation of cortical tissue from AD brains taken at autopsy demonstrate increased free radical production relative to non-AD controls. In addition, the activities of critical antioxidant enzymes, particularly catalase, are reduced in AD (Gsell et al., J. Neurochem. 64:1216-23, 1995), suggesting that the AD brain is vulnerable to increased ROS production. Thus, oxidative stress may contribute significantly to the pathology of mitochondria associated diseases such as AD, where mitochondrial dysfunction and/or elevated ROS may be present.
One hallmark pathology of AD is the death of selected neuronal populations in discrete regions of the brain. Cell death in AD is presumed to be apoptotic because signs of programmed cell death (PCD) are seen and indicators of active gliosis and necrosis are not found. (Smale et al., Exp. Neurolog. 133:225-230, 1995; Cotman et al., Molec. Neurobiol. 10:19-45, 1995.) The consequences of cell death in AD, neuronal and synaptic loss, are closely associated with the clinical diagnosis of AD and are highly correlated with the degree of dementia in AD (DeKosky et al., Ann. Neurology 27:457-464, 1990).
Mitochondrial dysfunction is thought to be critical in the cascade of events leading to apoptosis in various cell types (Kroemer et al., FASEB J. 9:1277-87, 1995), and may be a cause of apoptotic cell death in neurons of the AD brain. Altered mitochondrial physiology may be among the earliest events in PCD (Zamzami et al., J. Exp. Med. 182:367-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-72, 1995) and elevated reactive oxygen species (ROS) levels that result from such altered mitochondrial function may initiate the apoptotic cascade (Ausserer et al., Mol. Cell. Biol. 14:5032-42, 1994). In several cell types, including neurons, reduction in the mitochondrial membrane potential (xcex94xcexa8m) precedes the nuclear DNA degradation that accompanies apoptosis. In cell-free systems, mitochondrial, but not nuclear, enriched fractions are capable of inducing nuclear apoptosis (Newmeyer et al., Cell 70:353-64, 1994). Perturbation of mitochondrial respiratory activity leading to altered cellular metabolic states, such as elevated intracellular ROS, may occur in mitochondria associated diseases and may further induce pathogenetic events via apoptotic mechanisms.
Oxidatively stressed mitochondria may release a preformed soluble factor that can induce chromosomal condensation, an event preceding apoptosis (Marchetti et al., Cancer Res. 56:2033-38, 1996). In addition, members of the Bcl-2 family of anti-apoptosis gene products are located within the outer mitochondrial membrane (Monaghan et al., J. Histochem. Cytochem. 40:1819-25, 1992) and these proteins appear to protect membranes from oxidative stress (Korsmeyer et al, Biochim. Biophys. Act. 1271:63, 1995). Localization of Bcl-2 to this membrane appears to be indispensable for modulation of apoptosis (Nguyen et al., J. Biol. Chem. 269:16521-24, 1994). Thus, changes in mitochondrial physiology may be important mediators of apoptosis. To the extent that apoptotic cell death is a prominent feature of neuronal loss in AD, mitochondrial dysfunction may be critical to the progression of this disease and may also be a contributing factor in other mitochondria associated diseases.
Regardless of whether a defect underlying a disease associated with altered mitochondrial function may have mitochondrial or extramitochondrial origins, and regardless of whether a defect underlying altered mitochondrial function has been identified, the present invention provides methods that are useful for determining the risk or presence of diseases associated with such altered mitochondrial function, and for identifying agents that are suitable for treating such diseases. In particular, as is elaborated herein below, the present invention provides compositions and methods for the detection of diseases associated with altered mitochondrial function by quantification of unusual mtDNA-like sequences not found in mitochondria and referred to as extramitochondrial DNA (exmtDNA), and other related advantages.
Briefly stated, the present invention is directed to compositions and methods useful for detecting mitochondria associated diseases and involving extramitochondrial DNA (exmtDNA) sequences that are highly homologous to mitochondrial DNA (mtDNA). In one aspect the invention provides a method for determining the risk for or presence of a disease associated with altered mitochondrial function in a first subject suspected of having or being at risk for having such a disease, by comparing a ratio r for each of a first and a second biological sample containing extramitochondrial DNA and mitochondrial DNA, the first biological sample being obtained from the first subject and the second sample being obtained from a second subject known to be free of a risk or presence of a disease associated with altered mitochondrial function, using the formula:
r=x/(x+y)
wherein x is the amount of extramitochondrial DNA in a sample, and y is the amount of mitochondrial DNA in the sample; and therefrom determining the risk or presence of the disease. In an embodiment of the invention, the ratio r is calculated by a method that comprises contacting a biological sample containing extramitochondrial DNA and mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the extramitochondrial DNA and present in the mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA and to the mitochondrial DNA; and detecting hybridization of the primer to the extramitochondrial DNA and to the mitochondrial DNA, in order to therefrom quantify the extramitochondrial DNA and the mitochondrial DNA.
In another embodiment, the ratio r is calculated by a method comprising contacting a sample containing amplified extramitochondrial DNA and mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the amplified extramitochondrial DNA and present in the amplified mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA and to the mitochondrial DNA; and detecting hybridization of the primer to the extramitochondrial DNA and to the mitochondrial DNA, and therefrom quantifying the extramitochondrial DNA and the mitochondrial DNA. In another embodiment of this aspect of the invention the ratio r is calculated by a method comprising contacting a biological sample containing extramitochondrial DNA and mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the extramitochondrial DNA and present in the mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of said primer to the extramitochondrial DNA and to the mitochondrial DNA; and detecting hybridization and extension of the primer to the extramitochondrial DNA to produce a first product and hybridization and extension of the primer to the mitochondrial DNA to produce a second product distinguishable from the first product, and therefrom quantifying the extramitochondrial DNA and the mitochondrial DNA.
In another embodiment of this aspect of the invention the ratio r is calculated by a method comprising contacting a sample containing amplified extramitochondrial DNA and mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the amplified extramitochondrial DNA and present in the amplified mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA and to the mitochondrial DNA; and detecting hybridization and extension of the primer to the extramitochondrial DNA to produce a first product and hybridization and extension of the primer to the mitochondrial DNA to produce a second product distinguishable from the first product, and therefrom quantifying the extramitochondrial DNA and the mitochondrial DNA.
In another embodiment of this aspect of the invention the biological sample is treated by heating it in water to lyse cells contained in the sample, and then extracting cellular DNA from the lysed cells using an aqueous DNA extraction procedure. In certain embodiments of the invention the sample comprises a crude buffy coat fraction of whole blood. In certain other embodiments of the invention, the method further comprises the step of determining the ApoE genotype of the first subject and correlating said genotype with the risk or presence of disease. In some embodiments of the invention, the disease associated with altered mitochondrial function may be Alzheimer""s Disease, Huntington""s Disease, Parkinson""s Disease, dystonia, schizophrenia, non-insulin dependent diabetes mellitus, mitochondrial encephalopathy, lactic acidosis, and stroke, myoclonic epilepsy ragged red fiber syndrome, and Leber""s hereditary optic neuropathy.
Another aspect of the invention provides a method for quantifying extramitochondrial DNA, comprising: contacting a sample containing extramitochondrial DNA with an oligonucleotide primer having a nucleotide sequence complementary to at least a portion of the extramitochondrial DNA under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA; and detecting hybridization of the primer to the extramitochondrial DNA, and therefrom quantifying the extramitochondrial DNA.
It is another aspect of the invention to provide a method for quantifying extramitochondrial DNA, comprising: contacting a sample containing extramitochondrial DNA with an oligonucleotide primer having a nucleotide sequence complementary to at least a portion of the extramitochondrial DNA under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA; and detecting hybridization and extension of the primer to the extramitochondrial DNA to produce a product, and therefrom quantifying the extramitochondrial DNA.
Another aspect of the invention provides a method for quantifying extramitochondrial DNA, comprising: contacting a sample containing amplified extramitochondrial DNA with an oligonucleotide primer having a nucleotide sequence complementary to at least a portion of the extramitochondrial DNA under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA; and detecting hybridization of the primer to the extramitochondrial DNA, therefrom quantifying the extramitochondrial DNA.
In yet another aspect of the invention, a method is provided for quantifying extramitochondrial DNA by contacting a sample containing amplified extramitochondrial DNA with an oligonucleotide primer having a nucleotide sequence complementary to at least a portion of the extramitochondrial DNA under conditions and for a time sufficient to allow hybridization of the primer to the extramitochondrial DNA; and detecting hybridization and extension of the primer to the extramitochondrial DNA to produce a product, and therefrom quantifying the extramitochondrial DNA.
In one embodiment the extramitochondrial DNA is amplified by polymerase chain reaction, transcriptional amplification systems or self-sustained sequence replication. In certain embodiments of the various aspects of the invention, a single oligonucleotide primer is used. In certain embodiments of the invention a primer extension assay is used. In certain embodiments of the invention, the step of detecting may be by polymerase chain reaction, primer extension assay, ligase chain reaction or restriction fragment length polymorphism analysis.
In certain embodiments of the invention, the ratio r for a biological sample is calculated by determining the presence in the sample of a nucleotide sequence that may be SEQ ID NO:1, a portion of SEQ ID NO:1, SEQ ID NO:3 or a portion of SEQ ID NO:3. In one embodiment the portion of the nucleotide sequence of SEQ ID NO:1 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial cytochrome c oxidase. In another embodiment the portion of SEQ ID NO:1 corresponds to a portion of a mitochondrial cytochrome c oxidase encoding sequence that may be portion of a cytochrome c oxidase 1 (CO1) encoding sequence or a portion of a cytochrome c oxidase 2 (CO2) encoding sequence. In still other embodiments, the portion of the nucleotide sequence of SEQ ID NO:1 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial ATP synthetase subunit. In other embodiments, the portion of SEQ ID NO:1 corresponds to a portion of a mitochondrial ATP synthetase subunit encoding sequence that may be a portion of a sequence encoding ATP synthetase subunit 6 or a portion of a sequence encoding ATP synthetase subunit 8.
In some embodiments the nucleotide sequence of SEQ ID NO:1 corresponds to a portion of SEQ ID NO:2 that may be a portion of a sequence encoding ND1, a sequence encoding a portion of ND2 or a sequence encoding a portion of CO3. In other embodiments, the portion of the nucleotide sequence of SEQ ID NO:3 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial ATP synthetase subunit, which in some embodiments may further be a portion of a sequence encoding ATP synthetase subunit 6 or a portion of a sequence encoding ATP synthetase subunit 8. In still other embodiments, the nucleotide sequence of SEQ ID NO:1 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial tRNA, while in yet other embodiments the portion of the nucleotide sequence of SEQ ID NO:3 corresponds to a portion of the nucleotide sequence of SEQ ID NO:2 encoding a mitochondrial tRNA.
In another aspect the invention provides an isolated nucleic acid comprising all or a portion of the nucleotide sequence of SEQ ID NO:1 or a complementary sequence thereto. In another aspect the invention provides an isolated nucleic acid comprising all or a portion of a nucleotide sequence of SEQ ID NO:1 or a complementary sequence thereto, wherein the sequence of the isolated nucleic acid differs by at least one nucleotide from the corresponding sequence of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or a complementary sequence thereto. In another aspect the invention provides an isolated nucleic acid comprising all or a portion of the nucleotide sequence of SEQ ID NO:3 or a complementary sequence thereto. In another aspect the invention provides an isolated nucleic acid comprising all or a portion of a nucleotide sequence of SEQ ID NO:3 or a complementary sequence thereto, wherein the sequence of the isolated nucleic acid differs by at least one nucleotide from the corresponding sequence of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or a complementary sequence thereto.
In another aspect the invention provides a method for determining the risk or presence of a disease associated with altered mitochondrial function in a subject suspected of having or being at risk for having such a disease, by quantifying the amount of extramitochondrial DNA and the amount of mitochondrial DNA in a biological sample from the subject, and therefrom determining the risk or presence of the disease. It is another aspect of the invention to provide a method for determining the risk or presence of a disease associated with altered mitochondrial function in a first subject suspected of having or being at risk for having such a disease, by comparing the amount of extramitochondrial DNA and the amount of mitochondrial DNA in a biological sample from the first subject to the amount of extramitochondrial DNA and the amount of mitochondrial DNA in a biological sample from a second subject, and therefrom determining the risk or presence of the disease. In another aspect the invention provides a method for determining the risk or presence of a disease associated with altered mitochondrial function in a first subject suspected of having or being at risk for having such a disease, by quantifying the amount of extramitochondrial DNA and the amount of mitochondrial DNA in a biological sample from the subject and comparing the amount of extramitochondrial DNA and the amount of mitochondrial DNA to the amount of extramitochondrial DNA and the amount of mitochondrial DNA in a biological sample from a second subject known to be free of a risk or presence of a disease associated with altered mitochondrial function, and therefrom determining the risk or presence of the disease.
Another aspect of the invention provides a method of regulating a telomere by administering to a subject a nucleic acid molecule comprising all or a portion of SEQ ID NO:1 or a complementary portion thereto. In one embodiment, the administered nucleic acid molecule comprises an exmtDNA sequence. In another aspect, the invention provides a method of regulating a telomere by administering to a subject a nucleic acid molecule comprising all or a portion of SEQ ID NO:3 or a complementary portion thereto.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain aspects of this invention, and are therefore incorporated by reference in their entirety.